Scanning methods for three-dimensional imaging are known from the prior art, for example from C. Dunsby, “Optically sectioned imaging by oblique plane microscopy,” Opt. Express 16, 20306-20316 (2008) or WO 2015/109323 A2. An inclined-plane microscope is, for example, a so-called light sheet microscope for viewing tilted planes. Furthermore, the designation “inclined-plane microscope” also includes oblique plane illumination microscopes (OPM) and swept confocally-aligned planar excitation (SCAPE) microscopes, both of which are 3D scanning microscopes.
Inclined-plane microscopes operate with confocally-arranged planar excitation or illumination, which is scanned in a pivoting movement through a sample in the SCAPE microscope, whereas in OPM, the planar excitation is scanned linearly relatively to the sample, for example by a displaceable lens or a linear displacement of the sample.
In both representative techniques, a single optical element of high numerical aperture (NA), for example a lens with a high NA, is used, on the one hand, to realize the planar illumination of an illumination plane and, on the other hand, to capture the scattered light or fluorescent light emitted by the illuminated illumination plane via the same optical element and to provide it for imaging detection and data processing.
Produced thereby are image stacks consisting of images of the illumination planes which were respectively recorded at successive points in time and which are parallel to one another in the case of OPM and substantially parallel to one another in the case of SCAPE microscopy. The individual illumination planes are tilted relatively to the optical axis of the lens, which is used for illumination and imaging of the illuminated illumination planes.
The position of the various illumination planes is varied by a scanning element, for example a, preferably motorized, translation stage displaces a lens or lens arrangement or the sample (OPM) or by tilting a scanning mirror (SCAPE). The scanning element displaces or tilts both an illumination beam path and an observation beam path relatively to the examined sample.
If the user of an inclined-plane microscope wants to obtain a preview image of the examined sample in real time, this preview image for orientation in the sample can be obtained in that only the imaging of a single illuminated illumination plane in the sample is evaluated and reproduced, or a preview image is generated from the entire recorded image stack.
A disadvantage of the first procedure is that the preview image is tilted relatively to the optical axis of the lens, which makes the orientation in the sample more difficult for the user.
The disadvantage of the second approach is that the preview images cannot be delivered in real time and a movement of the sample by the user thus cannot be tracked instantaneously.